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Genetically modified foods
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C:\Documents And Settings\Louay Labban Uok\Desktop\All\Powerpoints\Gmo Slides 1
- 7. Which foods contain GM product? Sources: 1996-1999 Fernandez and McBride, 2000-2004: USDA, National Agriculture Statistics Service, Acreage.
- 8. Which foods yield viable plant DNA? Very Reliable Reliable Less Reliable Very Difficult / Not Possible Fresh corn Veggie sausages Veggie burgers Oil Fresh papaya Tortilla chips Fried corn snacks Salad dressing Corn bread mix Flavored tortilla chips Popcorn Cereal (eg cornflakes) Corn meal Puffed corn snacks Fries Wheat flour Soy flour Meatballs and burgers containing soy protein Potato chips Soy-based protein drinks/powders
- 10. How to test for GMOs ELISA : Test for presence of proteins expressed from genetic modifications Pro: Quick, cheap, low tech Con: Crop specific, protein stability PCR : Test for presence of inserted foreign DNA Pro: ID different GM crops, DNA stability Con: Expensive, timely
- 13. Why amplify a plant gene? To confirm that viable DNA was extracted and that negative GM result isn’t due to a non-viable template. Use highly conserved chloroplast gene from Photosystem II – part of the light reaction of photosynthesis.
- 14. Why use CaMV 35S and NOS ? CaMV 35S – Sequence for the promoter of 35S transcript of the Ca uliflower m osaic v irus. Used because it functions in every plant cell NOS- Sequence for no paline s ynthase terminator from soil bacterium Agrobacterium tumefacians Used because it evolved to be recognized in most plants
- 15. Extract DNA from food
- 17. Set up PCR reactions
- 18. Volumetric Measurements 50 μ l
- 20. PCR Review PCR Animation
- 21. The PCR Reaction How does it work? Heat ( 94 o C ) to denature DNA strands Cool ( 59 o C ) to anneal primers to template Warm ( 72 o C ) to activate Taq polymerase, which extends primers and replicates DNA Repeat 40 cycles
- 27. Clone the gene Ti plasmid ori Bt gene Bacillus thuringiensis Delta endotoxin crystal Ti genes
- 28. Engineer the gene Antibiotic resistance Ti plasmid ori Bt gene Ti genes STOP GO
- 29. Transform gene into plant Isolate plant cells Grow undifferentiated callus Transform cells Select cells Redifferentiate callus Grow transgenic plant
- 30. Backcross GM plant into high yield crops GM plant = yy GG High yield plant = YY gg YY gg x yy GG Y y G g YY gg x Y y G g YY g G Y yg G YY gg Y ygg YY g G x YY g G YY g G YY gg YYG g YYGG
- 31. 1 3 2 7 6 5 4 GMO positive GMO negative 7: PCR MW Ruler 1: non-GMO food with plant primers 2: non-GMO food with GMO primers 3: Test food with plant primers 4: Test food with GMO primers 5: GMO positive template with plant primers 6: GMO positive template with GMO primers Analysis of Results 1 3 2 7 6 5 4
Editor's Notes
- Bolded crops are widely grown & sold in US.
- 80% of soy crop in GMO
- ELISA used only for fresh food (cheaper, faster). Photosystem II gene that all plants have: use as test for viable plant DNA.
- Specifically the 32 kDa psbA gene
- About 65% of food crops use 35S promoter, by adding NOS detection, can detect about 80% of GM foods. 35S promoter drives expression of 35S RNA transcript of CaMV. 2 main transcripts of CaMV. 19S and 35S. 19S codes for proteins. 35S is reverse transcribed to make the virion DNA in the cytoplasm of the infected cell for the productions of new virus particles. Nopaline is an amino acid derivative, derived from arginine, produced in wound sites by tumor inducing (Ti) plasmid from A.tumefaciens.Nopaline provides food for A.tumefaciens and therefore gives selective advantage to A. tumefaciens. Nopaline also induces conjugation to transfer Ti plasmid into other bacteria. NOS genes are on the T-DNA that is transfered into the plant genome by the Ti plasmid
- Pest resistance = Bacillus thuringiensis (Bt), cry delta endotoxin (protein); Herbicide resistance = RoundUp Ready, glyphosate tolerance, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), from agrobacterium. Drought resistance = C4 plants, stomata that close to minimize water loss. Increased Nutritional Value = Golden Rice, beta carotene. Improved fruit = Flavr Savr tomatoes, delayed softening. Altered Ripening = malin (ethylene precursor; plant hormone).
- Maize line Bt11 was developed through a specific genetic modification to be resistant to attack by European corn borer (ECB; Ostrinia nubilalis ), a major insect pest of maize in agriculture. The novel variety produced the insecticidal protein, Cry1Ab, derived from Bacillus thuringiensis subsp. kurstaki (B.t.k.) HD-1 strain. Delta-endotoxins, such as the Cry1Ab protein expressed in Bt11, act by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry1Ab is insecticidal only to lepidopteran insects, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins. (www.agbios.com)
- Add promoter and terminator, streamline genes by removing introns. Ti plasmid from Agrobacterium.
- Take plant leaf sample, break up and develop callus on media. Callus is mass of undifferentiated cells. Transform using gene gun or other methods. Grow callus on selective media for transformants, transfer to growth media…becomes plant. Other methods, electroporation, agrobacterium, whiskers. Totipotent property of plants.
- Repeatedly backcross GM plant to high yield plant to reintroduce hybrid traits (genome).